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Abstract and IntroductionAbstract

Parkinsonian diseases comprise a heterogeneous group of neurodegenerative disorders, which show significant clinical andpathological overlap. Accurate diagnosis still largely relies on clinical acumen; pathological diagnosis remains the goldstandard. There is an urgent need for biomarkers to diagnose parkinsonian disorders, particularly in the early stages whendiagnosis is most difficult. In this review, several of the most promising cerebrospinal fluid candidate markers will bediscussed. Their strengths and limitations will be considered together with future developments in the field.

Introduction

Idiopathic Parkinson's disease (iPD) is a progressive neurological disorder initially described as a clinical entity by JamesParkinson and then embellished by Charcot and other nineteenth­century physicians, including Trousseau, Gowers and Erb.It is a clinical construct, based upon the presence of bradykinesia accompanied by at least one other characteristic feature,such as resting tremor, rigidity and impaired postural reflexes.[1] The signs and symptoms are usually asymmetrical at onsetand, typically, there is a good response to levodopa treatment.

'Parkinson­plus' or 'atypical parkinsonism' are terms that refer to a heterogeneous group of neurodegenerative disorders thatmay masquerade particularly in the early stages of the disease as Parkinson's disease (PD).[2] The 'plus' or 'atypical'descriptor indicates the presence of additional characteristics not usual in patients with iPD, such as early autonomicdisturbance and pyramidal signs exhibited by patients with multiple system atrophy (MSA), supranuclear gaze palsy andfrontal/dysexecutive syndrome by those with progressive supranuclear palsy (PSP), dystonia and myoclonus in corticobasaldegeneration (CBD) and early postural instability and falls by all of them. Another disease that could be classified as anatypical parkinsonian disorder is dementia with Lewy bodies (DLB), where dementia onset is before or within a year of onsetof extrapyramidal features. The earlier onset of dementia differentiates DLB from Parkinson's disease dementia (PDD).

Atypical parkinsonian disorders account for less than 10% of all parkinsonism and rarely respond with sustained improvementto levodopa. They usually follow a much more aggressive disease course than iPD and are characterised by atrophy toseveral different cortical and subcortical networks. Furthermore, atypical parkinsonism has been described in otherconditions, such as Alzheimer's disease (AD) and frontotemporal dementia (FTD).

Pathology

Protein misfolding and aggregation is seen with many neurodegenerative diseases. Based on pathological findings,parkinsonian syndromes are classified into α­synucleinopathies (PD, DLB and MSA) and primary tauopathies (PSP andCBD). For pathological lesions used in postmortem diagnosis of parkinsonism, see figure 1.

Cerebrospinal Fluid Biomarkers in ParkinsonianConditions: An Update and Future DirectionsNadia Magdalinou, Andrew J Lees, Henrik ZetterbergJ Neurol Neurosurg Psychiatry. 2014;85(10):1065­1075.

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Figure 1.

In Parkinson's disease (PD), there is loss of pigmented neurons from the substantia nigra and remaining neurons may bevery sparse (A). Lewy bodies can be observed in residual neurons (A, inset) and are highlighted, together with Lewy neuritis,using α­synuclein immunohistochemistry (B). Lewy bodies and Lewy neurites may be present in significant numbers in theneocortex (C, frontal cortex). In multiple system atrophy (MSA), α­synuclein is primarily deposited in the form of glialcytoplasmic inclusions in oligodendrocytes (D, putamen) and may also form inclusions in neuronal cytoplasm and nuclei(arrow) (E, pontine nuclei). In progressive supranuclear palsy tau forms, aggregates in neurons and glia, giving rise to tufted

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astrocytes (F, caudate) and neurofibrillary tangles (G, pontine nuclei). A characteristic feature of corticobasal degeneration(CBD) is the astrocytic plaque, formed from aggregated tau in the distal processes of astrocytes (H, parietal cortex). In CBD,tau also accumulates in neurons in the form of neurofibrillary tangles (H, inset a) and in oligodendrocytes as coiled bodies (H,inset b). (A) Haematoxylin and eosin; (B–D) α­synuclein immunohistochemistry; (F–H) tau immunohistochemistry. Bar in (A)represents 100 μm in (C); 50 μm in (A, D–G); 25 μm in inset A, B and H. Pathological images kindly provided by Dr JaniceHolton, Queen Square Brain Bank for Neurological Disorders, London.

α­Synuclein (α­Syn) has been found to be the major constituent of the intracellular aggregates in Lewy bodies and Lewyneurites (pathological hallmark of PD and DLB) and in the glial cytoplasmic inclusions in MSA.[3,4] The presence ofabnormally aggregated tau proteins in the form of neurofibrillary tangles, for example, are diagnostic of PSP.[5] Tau­positiveintracellular inclusions are the neuropathological findings in CBD.[6] Even though there are also neurofibrillary tangles in AD,Aβ plaques are closely tied to the primary disease process and thus AD is considered to be a secondary tauopathy. FTD canalso have underlying tau pathology.

There is often some overlap between syncleinopathies and tauopathies (for a review, see ref. 7). Co­occurrence of tau and α­Syn pathology has been found in neurons and oligodendrocytes in AD, PD and DLB.[8] α­Syn has complex and dynamicinteractions with tau. Each of these two proteins has the tendency to seed the aggregation of the other.[9] α­Syn inducesaggregation and polymerisation of tau, which promotes formation of intracellular amyloid­tau inclusions.[10] Similarinteractions have been described between α­Syn and Aβ pathology.[11]

Genetics

Recent advances in genetics have shed light on the underlying pathophysiology because mutations in the gene for eachmisfolded protein can give rise to an inherited form of a relevant neurodegenerative condition. For example, rare hereditaryforms of PD can be caused by mutations affecting the gene coding for α­Syn (SNCA); PARK1 (missense) and PARK4(duplication, triplication).[12] Furthermore, in both PD and to a lesser extent in MSA, population studies demonstrated anassociation between disease risk and distinct single­nucleotide polymorphisms in SNCA. DJ­1(PARK7) mutations can lead torare forms of autosomal­recessive PD, pointing towards mitochondrial damage/oxidative stress pathways drivenpathogenesis.[13] Even though PD is not a 'tauopathy', population studies also showed variants in tau (MAPT) gene,particularly the H1 haplotype, as another risk factor for PD (for a review, see ref. 14). Several tauopathies are associated withvariants in MAPT, including CBD, FTD linked to chromosome 17 (FTDP­17T) and PSP.[15] The fact that the MAPT/tauhaplotype also shows an association with PD strongly suggests that the pathogenic cascades in the tauopathies may berelated to those in the synucleinopathies.[16]

Diagnostic Challenges

Accurate diagnosis of parkinsonian disorders still relies heavily on clinical acumen, although imaging and ancillaryinvestigations may be helpful in some situations. In one postmortem series, 24% of patients clinically diagnosed withidiopathic PD by a consultant neurologist during life were found to have an alternative diagnosis.[1]

Cerebrospinal Fluid Biomarkers

A biomarker is "a characteristic that is objectively measured and evaluated as an indicator of normal biological processes,pathogenic processes or pharmacologic response to a therapeutic intervention".[17] An 'ideal' biomarker should be sensitive,reproducible, closely associated with the disease process, non­invasive and inexpensive.

Cerebrospinal fluid (CSF) has more physical contact with the brain than any other fluid and as such represents a potentiallyreliable biomarker source. Unlike plasma, CSF is not separated from the brain by the tightly regulated blood–brain barrier.Proteins/peptides that may be directly reflective of brain specific activities or disease pathology would most likely diffuse intothe CSF. Furthermore, CSF can be tested serially, which makes possible the study of protein changes reflecting the evolvingpathology throughout the clinical course of the disease. This is preferable to pathological studies, which only reveal theterminal changes of a disease process that has developed over decades.

Historical Background

CSF has been widely investigated in parkinsonian disorders and is considered to offer the most promising insights into thedisease process. Historically, because of dopaminergic abnormalities in parkinsonism, the first compounds to be tested aspotential markers were dopamine and other monoamines and their metabolites. In the 1960s and 1970s, reduced CSFmonoamine concentrations (homovanillic acid and 5­hydroxyindoleacetic acid) were found in patients with parkinsonism and

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dementia.[18] A study conducted at the National Hospital, Queen Square, London, assessed the effect of levodopa treatmentin CSF homovanillic acid concentration of PD patients. Before levodopa treatment, homovanillic acid concentration was low inall patients, while after treatment it rose to a level that correlated significantly with the levodopa dose.[19]

As these metabolic results were prone to be influenced by a multitude of other factors,[7] the quest went further to investigatea priori defined compounds, such as α­Syn and tau. These were tested in patients and in healthy controls, looking fordifferences, patterns and associations. Even though several promising candidates exist, there is still no reliable biomarker.

Methods

We reviewed the potential use of CSF proteins as biomarkers in parkinsonism, focusing on α­Syn, neuronal injury markersand Aβ42. In addition, we briefly reviewed the latest novel markers and the 'omics' approach. We performed aPubMed/Medline search and limited searches to studies reported in English and published after 2006, including antemortem,human, lumbar CSF; all studies included at least one parkinsonian cohort compared with healthy or neurological controls. Wecombined searches with 'Parkinson's disease', 'progressive supranuclear palsy', 'multiple system atrophy', 'corticobasalsyndrome' (CBS), 'corticobasal degeneration', 'Parkinson's disease dementia', 'dementia with Lewy bodies', 'Lewy bodydementia', 'parkinsonism', 'synucleinopathies', 'tauopathies', 'neurodegenerative diseases' with 'CSF biomarkers' and specificbiomarkers ('α­Syn', 'tau', 'phosphorylated tau', 'Aβ42', 'neurofilaments', 'neuronal injury markers', 'inflammatory', 'metabolic'and 'oxidative stress markers'). Further references were found manually from identified publications. For a review of theearlier literature, not captured using the time limit of our search criteria, see Eller and Williams.[20]

CSF Biomarker Candidates in ParkinsonismAβ42

Aβ42 is a 42 amino­acid long, aggregation­prone protein, derived from the proteolytic processing of amyloid precursor proteinand is a major component of neuritic plaques in AD. Cognitive impairment and dementia are much more common inparkinsonism than in the general population and have a detrimental effect on quality of life and life expectancy. The linkbetween Aβ42 and PD and dementia has been studied extensively (see ).

Table 1. CSF Aβ42 in parkinsonian disorders

Researchgroups

Participants Main findings

Kang et al 21 PD n=39 (drug­naïve patients), HC n=63 Decrease in PD vs HC

Compta et al 31Baseline: PD n=27 (non­demented)

18 month follow­up: PD n=16 (non­demented), PD n=11(dementia converters)

Decrease in dementia converters

Bech et al 32PD n=22, PDD n=3, DLB n=11, MSA n=10, PSP n=20, CBDn=3

Decrease in DLB vs other diseasegroups

Hall et al 26PD n=90, PDD n=33, DLB n=70, PSP n=45, CBD n=12, MSAn=48, AD n=48, controls n=107

Decrease in AD>DLB+PDD

Schoonenboomet al 33

DLB n=52, PSP n=20, CBD n=16, AD n=512, FTD n=144, VaDn=34, CJD n=6, controls n=275

Decrease in AD>VaD>DLB>CBD

Parnetti et al 27 PD n=38, DLB n=32, AD n=48, FTD n=31, controls n=32

Decrease in AD, FTD+DLBvs PD and controls

No difference between PDand controls

Andersson et al34 DLB n=47, PDD n=17, AD n=150 Decrease in DLB vs PDD

Shi et al 22

Discovery cohort: PD n=126, MSA n=32, AD n=50, controlsn=137 Slight decrease in PD and MSA vs

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Validation cohort: PD n=83controls

Montine et al 28 PD n=41, PDD n=11, AD n=49, HC n=150 Decrease in PDD vs HC

Süssmuth et al29

PSP­RS n=20, PSP­P n=7, MSA­P n=11, MSA­C n=14, PDn=23, controls n=20

No difference inparkinsonian syndromes

Lower in PSP­RS vs PSP­P

Alves et al 23 PD n=109, AD n=20, HC n=36 Decrease in PD vs HC

Ohrfelt et al 30 PD n=15, DLB n=15, AD n=66, controls n=55Decrease in AD+DLB vs controlsand PD

Compta et al 24 PD n=20, PDD n=20, HC n=15 Decrease in PDD>PD vs HC

Parnetti et al 25 PD n=20, PDD n=8, DLB n=19, AD n=23, HC n=20 Decrease in DLB>PDD>PD

AD, Alzheimer's disease; CBD, corticobasal degeneration; CJD, Creutzfeldt–Jakob disease; CSF, cerebrospinal fluid; DLB,dementia with Lewy bodies; FTD, frontotemporal dementia; HC, healthy controls; MSA, multiple system atrophy; MSA­C,multiple system atrophy cerebellar type; MSA­P, multiple system atrophy parkinsonian type; PD, Parkinson's disease; PDD,Parkinson's disease dementia; PSP, progressive supranuclear palsy; PSP­P, progressive supranuclear palsy–parkinsonism;PSP­RS, progressive supranuclear palsy–Richardson's syndrome; VaD, vascular dementia.

In most studies, Aβ42 is significantly reduced in PD compared with controls and is associated with worse cognitiveperformance.[21–25] However, other investigations showed no difference between PD and controls.[26–30]

Compta et al [31] collected CSF from 27 non­demented PD patients and followed them over time. Patients who converted todementia within 18 months had a significantly lower baseline CSF Aβ42 than the patients who remained non­demented.

DLB patients have the lowest CSF levels of Aβ42 among the parkinsonian cohorts.[25,32–34] One study found that almost halfof DLB patients had a CSF biomarker profile consistent with AD,[33] which agrees with the knowledge of Aβ pathology in thisdisease.[35–37]

There is evidence that low Aβ42, a marker of Aβ plaque pathology, may predict cognitive decline in patients with PD,[38] butother longitudinal studies with larger cohorts are necessary to clarify this further.

α­Syn

α­Syn is a 140 amino­acid long protein that localises to presynaptic terminals and is widespread in the brain, comprising 1%of cytosolic protein. In presynaptic terminals, α­Syn is present in close proximity to the synaptic vesicles. The precise functionof α­Syn is obscure, but it is speculated that its main role is in the control of neurotransmitter release.[39] Although mostlyconsidered an intracellular protein, α­Syn is capable of transfer between cells leading to a speculation of a prion­likemechanism operating in PD pathology spread.[40]

α­Syn can be modified by truncation, acetylation, phosphorylation, oxidation, nitrosylation, glycation or glycosylation.[41] Lewybodies are formed mostly of post­translationally modified α­Syn. α­Syn deposition is key in the pathogenesis ofsynucleinopathies. In vitro, similar to AD, α­Syn fibrillation involves α­Syn oligomerisation followed by oligomer conversioninto mature amyloid cultures, which are toxic to cultured neuronal cells.[42]

Total α­Syn (t­α­Syn)

Inconsistent results were initially reported in parkinsonian conditions with studies demonstrating considerable overlap of t­α­Syn in several neurodegenerative conditions.[30,43–45] A consensus is now emerging, and the vast majority of recent studies(predominantly using ELISA techniques) have shown a reduction of t­α­Syn levels in PD compared with controls.[21,22,26,46–55] In addition, there is decreased t­α­Syn in other synucleinopathies, such as MSA and DLB,[26,47,49,50,51,54] without gooddiscriminatory value between the groups (see ).

Table 2. CSF α­synuclein in parkinsonian disorders

ResearchParticipants Analyte Method Main findings

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groups

Van Dijk etal 46

PD n=53, HC n=50

t­α­Syn

t­α­Syn/t­proteinratio

TR­FRETDecrease in both t­α­Syn+t­α­Syn/t­protein ratio levels in PD vsHC

Kang et al201321

PD n=39 (drug­naïve patients),HC n=63

t­α­Syn ELISA Decrease in PD vs HC

Wennströmet al 47

PD n=38, PDD n=22, DLB n=33,AD n=46, HC n=52

t­α­Syn ELISADecrease in PDD > PD > DLB vsAD+HC

Mollenhaueret al 48

PD n=78 (de novo, drug­naïvepatients), HC n=48

t­α­Syn ELISA (3rd generation)Decrease in de novo PD patientsvs HC

Hall et al 26PD n=90, PDD n=33, DLB n=70,PSP n=45, CBD n=12, MSAn=48, AD n=48, controls n=107

t­α­SynBead­based multi­analyte assay(Luminex)

Modest decrease in AD >DLB+PDD > PD+MSA vscontrols, AD and PSP

Aerts et al43

PD n=58, MSA n=47, DLB n=3,VaD n=22, PSP n=10, CBD n=2

t­α­Syn ELISA No difference between groups

Tateno et al49

PD n=11, DLB n=6, MSA n=11,AD n=9, controls n=11

t­α­Syn ELISA

Decrease in PD, DLB,MSA vs AD+ controls

No difference among PD,DLB, MSA

Wang et al50

Discovery cohort:

PD n=83, MSA n=14, PSD n=30,AD n=25, HC n=51

Validation cohort:

PD n=109, MSA n=20, PSPn=22, AD n=50, HC n=71

t­α­Syn

p­α­Syn

p­α­Syn:t­α­Synratio

Bead­based multi­analyte assay(Luminex)

t­α­Syn decrease inPD+MSA vs controls

Increase α­Syn ratio inMSA vs PSP

Increase α­Syn ratio in PDvs controls and PSP

Park et al 58PD (drug­naïve) n=23, controlsn=18

t­α­Syn

o­α­Syn

Dual ELISA method forsimultaneousmeasurement of t­ando­α­Syn

t­α­Syn: no difference

o­α­Syn: increase in PD

Mollenhaueret al 54

Training cohort: PD n=51, DLBn=55, MSA n=29, AD n=62,controls n=76

Validation cohort: PD n=273,DLB n=66, PSP n=8, MSA n=15,NPH n=22, controls n=23

t­α­SynELISA (1st and 2ndgeneration)

Decrease in PD, DLB,MSA vs AD, NPH, PSPand controls

High degree ofconcordance in t­α­Synlevels between PD+MSA

Parnetti etal 27

PD n=38, DLB n=32, AD n=48,FTD n=31, controls n=32

t­α­Syn

t­α­Syn/t­tau

ELISA

t­α­Syn decrease in alldiseased groups(especially DLB/FTD)

Ratio: decrease in PD vsall other diseased groups

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ratio

Shi et al 22

Discovery cohort: PD n=126,MSA n=32

AD n=50, controls n=137

Validation cohort :PD n=83

t­α­SynBead­based multi­analyte assay(Luminex)

Decrease in PD vs controls andAD

Tokuda et al57

First cohort (all analytes):

PD n=32, controls n=28

Second cohort (o­asyn):

PD n=25, AD n=35, PSP n=18,controls n=43

t­α­Syn

o­α­Syn

o­α­Syn:t­α­Synratio

ELISA

t­α­Syn: trend towardsdecrease in PD

o­α­Syn+ratio increase inPD

Hong et al52 PD n=117, AD n=50, HC n=132 t­α­Syn

Bead­based multi­analyte assay(Luminex)

Decrease in PD vs AD andcontrols (after omitting sampleswith high haemoglobinconcentration)

Noguchi­Shinoharaet al 44

DLB n=16, AD n=21 t­α­Syn ELISA No difference

Spies et al45

DLB n=40, AD n=131, VaDn=28, FTD n=39

t­α­Syn ELISA No difference

Ohrfelt et al30

PD n=15, DLB n=15, AD n=66,controls n=55

t­α­Syn ELISADecrease in AD, no difference inparkinsonian groups

Mollenhaueret al 53

PD n=8, DLB n=38, AD n=13,CJD n=8, controls n=13

t­α­SynELISA (1st and 2ndgeneration)

Marginal decrease in LBD andPD vs all other groups

Tokuda et al55 PD n=38, controls n=38 t­α­Syn ELISA Decrease in PD vs controls

AD, Alzheimer's disease; CBD, corticobasal degeneration; CJD, Creutzfeldt–Jakob disease; CSF, cerebrospinal fluid; DLB,dementia with Lewy bodies; FTD, frontotemporal dementia; HC, healthy controls; MSA, multiple system atrophy; NPH, normalpressure hydrocephalus; PD, Parkinson's disease; PDD, Parkinson's disease dementia; PSP, progressive supranuclearpalsy; TR­FRET, time­resolved Förster's resonance energy transfer; VaD, vascular dementia.

Mollenhauer et al assessed levels of t­α­Syn in patients with synucleinopathies, patients with tauopathies and in neurologicalcontrols without neurodegenerative disease, first in a training set and then in a validation set. In the training set, acombination of t­α­Syn, t­tau and age differentiated synucleinopathies from neurological controls and AD with an area underthe curve (AUC) of 0.908. Only t­α­Syn levels and not t­tau or Aβ42 discriminated PD and MSA from controls with a positivepredictive value of 91%.[54]

Parnetti et al investigated whether the combination of t­tau, p­tau and t­α­Syn can improve differentiation of PD from DLB,AD, FTD and controls. They found an inverse correlation between t­α­Syn and total tau in all subjects and a lack of specificityof CSF t­α­Syn determination alone as a marker of synucleinopathy (sensitivity 94%, specificity 25%). However, t­tau/t­α­Synand p­tau/t­α­Syn ratios were identified as possible biomarkers for PD (sensitivity 89%, specificity 61%).[27]

Shi et al [22] also showed that a combination of t­α­Syn and p­tau/t­tau could discriminate PD from MSA with a sensitivity of90% and a specificity of 71%, when blood contaminated samples were excluded. t­α­Syn was decreased in PD andespecially in MSA compared with controls.

In most studies, there was no correlation of t­α­Syn with disease duration or disease severity. Interestingly, gender­specificvariations were reported in levels of t­α­Syn.[47] Both Mollenhauer et al [48] and Kang et al [21] studied drug­naïve PD patientsand still found reduction in t­α­Syn, so it was proven that this finding was not related to a dopaminergic medication effect.

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There are several theories why there is reduced t­α­Syn in PD, MSA and DLB. High brain levels of pathological t­α­Syn andlow CSF levels may reflect a reduction of 'free' t­α­Syn circulating in the CSF. This could be similar to 'pathological proteintrapping' reported for brain Aβ42 in AD CSF.[56]

Oligomeric and Phosphorylated α­Syn

Tokuda et al evaluated soluble α­Syn oligomers as potential early markers of PD and found that both the level of oligomericα­Syn and the oligomer/t­α­Syn ratio were substantially higher in patients with PD (including those with mild and early­stagedisease) compared with healthy controls and patients with non­neurodegenerative neurological conditions. CSF oligomer/t­α­Syn ratio had a sensitivity of 89.3% and a specificity of 90.6% for PD.[57] These findings were replicated in two further,independent studies.[58,59] Both oligomeric and phosphorylated oligomeric forms of α­Syn were detected in postmortemventricular CSF, which may be useful in distinguishing between PD, DLB and MSA.[60] The results need to be replicated inlarger groups of living patients.

Neuronal Injury MarkersTau

Tau is important for the function of axonal microtubules and, as a result, plays an important role in the structural integrity ofthe neuron and axonal support. When hyperphosphorylated, it has reduced binding affinity for microtubules, causing theirmalfunction. At the same time, it adopts an abnormal conformation leading to aggregation and inclusion formation.[61]

Total and Phosphorylated Tau (t­tau and p­tau)

In the past, there were inconclusive results when assessing tau levels in CSF of parkinsonian patients (see ). In PD, moststudies found normal values,[23,24,25,26,27,29,30] but lower levels were also reported.[21,22,28] In atypical parkinsonism, high t­tau levels were found in DLB[25,27,34] and low p­tau/t­tau ratio in MSA and PSP compared with PD.[29] However, otherinvestigations found no difference between parkinsonian syndromes.[22,26,30] In particular, no significant change has beenseen in PSP.[26] Age, not diagnosis, is thought to be the strongest factor affecting t­tau protein levels.[54]

Table 3. CSF neuronal injury markers: tau, neurofilament light chain (NF­L) and glial fibrillary acidic protein (GFAP) inparkinsonian disorders

Researchgroups

Participants Analyte Method Main findings

Kang et al 21PD n=39 (drug­naïve patients), HCn=63

t­tau, p­tau

Bead­based multi­analyte assay(Luminex)

Decrease in t­tau+p­tau in PDvs controls

Luk et al 64PDD n=11, PSP n=44, CBS n=22,AD n=11, controls n=34

3R/4Risoforms

Immuno­PCR(adapted fromsandwich ELISAs)

Decrease in 4R­tau inPSP and AD vs controls

Lower 4R­tau in AD vsPDD

­No difference in 3R­tau

Hall et al201226

PD n=90, PDD n=33, DLB n=70,PSP n=45, CBD n=12, MSA n=48,AD n=48, controls n=107

t­tau, p­tau

NF­L

Bead­based multi­analyte assay(Luminex)

Increased t­and p­tau inAD vs DLB+PDD

NF­L differentiates PDfrom atypicalparkinsonism

Bech et al 32PD n=22, PDD n=3, DLB n=11,MSA n=10, PSP n=20, CBD n=3

NF­L ELISA

Higher NF­L levels inatypical parkinsoniandisorders vs PD

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No difference betweenparkinsonian groups

Andersson etal 34

DLB n=47, PDD n=17, AD n=150t­tau, p­tau

ELISA Increased t­tau in DLB vs PDD

Shi et al 22Discovery cohort: PD n=126, MSAn=32, AD n=50, controls n=137

Validation cohort: PD n=83

t­tau, p­tau

Bead­based multi­analyte assay(Luminex)

­Decrease in PD vs tocontrols

­Decrease in PD+MSAvs AD

Parnetti et al201127

PD n=38, DLB n=32, AD n=48,FTD n=31, controls n=32

t­tau, p­tau

ELISA

Increase inAD>FTD>DLB vs PDand controls

No difference betweenPD and controls

Kuiperij et al102 NA

33/55

kDa tauforms

Immunoprecipitationassay and westernblotting

Not able to detect tauform ratio

Suggested that 33/55kDa bands seen areheavy and light IgGchains

Borroni et al103

PSP n=18, CBS n=16, FTD n=28,controls n=25

33/55

kDa tauforms

Immunoprecipitationassay and westernblotting

tau form ratio significantlyreduced in PSP vs othergroups

Constantinescuet al 71

PD n=10, MSA n=21, PSP n=14,CBD n=11, HC n=59

(×2 consecutive samples)

NF­L

GFAPELISA

NF­L: normal levels inPD, elevated in MSA,PSP+CBD

No statisticalsignificance overtime

GFAP: no difference

Montine et al28

PD n=41, PDD n=11, AD n=49,HC n=150

t­tau, p­tau

Bead­based multi­analyte assay(Luminex)

t­tau: no differencebetween parkinsoniangroups

p­tau: reduced in PD vsHC

Süssmuth et al29

PSP­RS n=20, PSP­P n=7, MSA­P n=11, MSA­C n=14, PD n=23,controls n=20

t­tau, p­tau

GFAP

ELISA

p­tau/t­tau ratio lower inPSP and MSA vs PD

GFAP: increase inparkinsonian syndromes

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(no difference betweendisease groups)

Alves et al 23 PD n=109, AD n=20, HC n=36t­tau, p­tau

ELISANo difference between PD andcontrols

Ohrfelt et al 30PD n=15, DLB n=15, AD n=66,controls n=55

t­tau, p­tau

ELISANo difference betweenparkinsonian groups

Compta et al 24 PD n=20, PDD n=20, HC n=15t­tau, p­tau

ELISAt­ and p­ tau: increase in PDDvs PD and controls

Parnetti et al 25PD n=20, PDD n=8, DLB n=19,AD n=23, HC n=20

t­tau, p­tau

ELISA

t­tau: DLB > PDD >controls

p­tau: no differencebetween parkinsoniangroups

Borroni et al 65PSP n=21, CBS n=20, FTD n=44,AD n=15, PD n=10, DLB n=15,controls n=27

33/55

kDa tauforms

Semiquantitativeimmunoprecipitationand western blotting

tau forms significantly reducedin PSP vs controls and otherneurodegenerative diseases

Brettschneideret al 70

PD n=22, MSA n=21, PSP n=21,CBD n=6, controls n=45

NF­H ELISAIncreased in MSA and PSP vsPD, CBD and controls

AD, Alzheimer's disease; CBD, corticobasal degeneration; CBS, corticobasal syndrome; CSF, cerebrospinal fluid; DLB,dementia with Lewy bodies; FTD, frontotemporal dementia; HC, healthy controls; MSA, multiple system atrophy; MSA­C,multiple system atrophy cerebellar type; MSA­P, multiple system atrophy parkinsonian type; NF_H, neurofilament heavychain; NF­L, neurofilament light chain; PD, Parkinson's disease; PDD, Parkinson's disease dementia; PSP, progressivesupranuclear palsy; PSP­P, progressive supranuclear palsy–parkinsonism; PSP­RS, progressive supranuclear palsy–Richardson's syndrome.

t­tau and p­tau may prove useful in differentiating AD from PD and can perhaps improve diagnostic accuracy when used incombination with other markers rather than on their own.

Tau Isoforms

Imbalances in the homeostasis of tau isoforms with three­ (3R­tau) and four­ (4R­tau) microtubule­binding repeat domainsare important in neurodegenerative disease pathogenesis. In a normal adult brain, there are comparable levels of 3R­ and4R­[62] but in PSP, CBD and FTDP­17 cases, the neurofibrillary tangles and glial inclusions are predominantly 4R, whereasPick bodies in FTD are predominantly 3R­tau[63] and neurofibrillary tangles in AD contain both 3R­ and 4R­tau isoforms.

Luk and colleagues had previously developed antibodies selective for the two isoforms and adapted an immuno­PCRprocedure in order to detect the isoforms' miniscule amounts in the CSF. Decrease in 4R­tau isoform was found in PSP andAD compared with CBS, PDD and controls. There was no difference in 3R­tau.[64]

We think that 4R­tau could be used as a marker of disease progression in PSP, but further large samples and longitudinalseries are needed.

Truncated Tau Forms

Borroni and colleagues looked at full­length (55 kDa) and truncated (33 kDa) tau forms in several neurodegenerativediseases. In ratio with the full­length tau forms, the truncated tau forms (33 kDa/55 kDa forms) were substantially reduced inPSP compared with healthy controls (sensitivity 96% and specificity 85% PSP compared with PD/DLB; sensitivity 90% andspecificity 76.2% PSP compared with CBD).[65] These fragments are proteolytic products of tau that were detected byimmunoprecipitation techniques, which are more time consuming, less quantitative and more operator­dependent than ELISAtechniques.

Findings were reproduced by the same group in another cohort of patients.[66] However, these results were not reproduced

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by another group, which did not find a reduced tau ratio in an independent cohort of PSP patients,[67] speculating that the33/55 kDa bands seen are heavy and light IgG chains. Recent findings of other endogenous tau fragments in CSF suggestthat specific assays for these fragments should be developed and evaluated in relation to different tauopathies.[68]

Neurofilament Light Chain Protein (NF­L)

Neurofilaments are major structural elements, whose main role is to maintain the axonal calibre and neuronal shape and size.[69] They are, thus, critical for the morphological integrity of neurons and for the conduction of nerve impulses along axons.They are composed of three subunits of different molecular weights: light, medium and heavy chain.

Neurofilament heavy chain (NF­H) forms an important component of the cytoskeleton. Higher CSF levels of NF­H were foundin PSP and MSA compared with PD, CBD and neurological controls.[70]

Neurofilament light chain forms the backbone of neurofilaments and can self­assemble. Increased levels in CSF reflectaxonal degeneration of large myelinated axons. Recent studies showed consistent results in differentiating PD from atypicalparkinsonian conditions[26,32,71] but not in discriminating between atypical parkinsonian syndromes. Consecutive analyses ofCSF showed no increase in NF­L levels with disease progression.[71]

NF­L can be useful in the differential diagnosis of PD versus other neurodegenerative conditions as it is very sensitive indetecting more aggressive neuronal death than occurs in PD.

Glial Fibrillary Acidic Protein

Glial fibrillary acidic protein (GFAP) is a protein predominantly expressed in fibrillary astrocytes. Disintegration of astroglialcells postacute brain injury can lead to high CSF GFAP levels. Süssmuth et al [29] showed that there are increased levels inparkinsonian syndromes compared with controls (patients with other neurological disorders), but there was no differencebetween diseased groups. However, another group found similar GFAP levels in parkinsonian syndromes and healthycontrols without significant change over time.[71]

Other Candidate MarkersOxidative Stress Markers

DJ­1. DJ­1 is a multifunctional protein involved in many processes. It is thought to have a protective role in oxidative stressduring neurodegeneration (). As we have already discussed, it has been linked to autosomal­recessive PD. Results on DJ­1as a CSF biomarker have been inconsistent so far. One study showed decreased levels in PD compared with controls with asensitivity of 90% and a specificity of 70%,[52] whereas another showed no difference among parkinsonian syndromes[72] andthe most recent one demonstrated significant increase in MSA compared with PD and controls.[73] The diagnostic accuracyfor discriminating MSA from PD was improved by combining DJ­1 levels with t­tau and p­tau levels.

Table 4. CSF biomarkers for oxidative stress, inflammation and energy failure in parkinsonian disorders

Researchgroups

Participants Analyte Method Main findings

Herbert et al 73PD n=43, MSA n=23,controls n=30

DJ­1 ELISA

Increase inMSA>PD

Significantdifference in MSAvs PD, MSA vscontrols and PDvs controls

Constantinescuet al 78

PD n=6, MSA n=13, PSPn=18, CBD n=6, HC n=18

UrateEnzymatic method on amodular system

No difference

Lowest levels inDLB, but nodifference

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Wennstrom etal 47

PD n=38, PDD n=22, DLBn=33, AD n=46, HC n=52

Neurosin ELISAbetweensynucleinopathies

When pooled,synucleinopathiesdecrease levelsvs AD+HC

Goldstein et al82

PD n=34, MSA n=54, PAFn=20, HC n=38

Dihydroxyphenylaticacid (DOPAC)

Batch aluminaextraction followed byliquid chromatographywith electrochemicaldetection

Decrease in PD,MSA and to alesser degreePAF vs HC

No differencebetweensynucleinopathygroups

Salvesen et al72

PD n=30, DLB n=17, MSAn=14, PSP n=19

DJ­1 ELISANo difference amonggroups

Maetzler et al77

PD n=55, PDD n=20, DLBn=20, controls n=76

Uric acidADVIAanalyser+photometricmethods

Increase in PD vs DLB

Shi et al 22Discovery cohort: PDn=126, MSA n=32 ADn=50, controls n=137Validation cohort: PD n=83

DJ­1 FractalkineBead­based multi­analyte assay(Luminex)

DJ1: decrease inMSA+PD vscontrols+AD

Fractalkine:decrease in MSAvs PD,AD+controls

LeWitt et al 81PD n=217 (samplescollected ×2 occasions)HC n=26

Homovallinicacid/xanthine ratio

Gas chromatography­mass spectrometry

Increased ratio inPD vs HC

Ratio increasedfurther in PDspecimenscollected up to 2years later

Wang et al 79PD n=86, MSA n=20, ADn=38 HC n=91

ComplementC3/factor H (FH)

Bead­based multi­analyte assay(Luminex)

C3: decrease inMSA vs PD+HC;increase in AD vsall other groups

FH: increase inAD vs PD+HC

C3/FH ratio:decrease in MSAvs all othergroups

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Maetzler et al80

PD n=38, PDD n=20, DLBn=21 m, controls n=23

Neprilysin Fluorometric assayDecrease in DLB+PDDvs PD+ controls

Hong et al 52PD n=117, AD n=50, HCn=132

DJ­1Bead­based multi­analyte assay(Luminex)

Decreased levelsin PD vs Controlsand AD

No differencebetween AD+controls

AD, Alzheimer's disease; CBD, corticobasal degeneration; CSF, cerebrospinal fluid; DLB, dementia with Lewy bodies; HC,healthy controls; MSA, multiple system atrophy; PAF, pure autonomic failure; PD, Parkinson's disease; PDD, Parkinson'sdisease dementia; PSP, progressive supranuclear palsy.

8­Hydroxydeoxyguanosine (8­OHdG). 8­OHdG is a marker of oxidation and mitochondrial dysfunction inneurodegeneration and malignancy. CSF 8­OHdG levels were increased in non­demented PD patients compared withcontrols and there was a negative correlation with MMSE levels in PDD.[74]

Urate. Urate is an endogenous and most potent antioxidant. Even though there is considerable evidence linking low serumlevels of urate to PD,[75,76] CSF studies have shown inconsistent results. Maetzler et al [77] found increase levels in PDcompared with DLB, but Constantinescu et al [78] showed no difference among parkinsonian groups and healthy controls.

Inflammatory Markers

Fractalkine. Fractalkine is an inflammatory cytokine that acts as a neurotrophic and antiapoptotic factor in the centralnervous system. It was decreased in MSA and could alone differentiate between PD and MSA with a sensitivity of 99% and aspecificity of 95%.[22] In addition, the fractalkine/Aβ42 ratio was closely associated with disease severity and progression inPD. These results are in need of replication.

Complement C3/Factor H Ratio. The C3/factor H ratio in CSF was significantly decreased in MSA compared with PD, ADand healthy controls. Increased levels of C3 or factor H, together with decreased levels of Aβ42, correlate positively withdisease severity and progression in PD.[79]

Neurosin. Neurosin is a protein expressed in human brain tissue, and it is one of several enzymes suggested to cleave α­Syn. A study comparing neurosin levels in synucleinopathies showed lowest levels in DLB, but no difference among DLB,PDD and PD. However, when pooled together, synucleinopathies had significantly lower neurosin levels compared with ADand controls.[47]

Neprilysin. Neprilysin is a membrane bound presynaptic protein involved in Aβ clearance. CSF levels were significantlydecreased in DLB and PDD compared with PD and controls, and they correlated well with Aβ42 levels in all cohorts.[80]

Catecholamine Metabolites

Homovanillic Acid (HVA)/Xanthine Ratio. HVA is the major catabolite of dopamine and has been extensively studied in thepast in relation to PD, as described above. Xanthine is the immediate precursor of urate. HVA/xanthine ratio was increased inPD compared with controls and correlated with diseased severity.[81]

Dihydroxyphenylatic aCid (DOPAC). Depletion of dopamine (a catecholamine) in basal ganglia is a defining neurochemicalcharacteristic in PD. DOPAC is a neuronal metabolite of catecholamines. It was found to be decreased in PD and MSAcompared with healthy controls, but there was no difference between synucleinopathy groups.[82]

The above compounds may be promising candidate makers, but they need verification in further studies. CSF HVA has beenextensively studied in relation to PD and treatment response but still has no definite place in the clinical routine.

Lysosomal Dysfunction

Lysosomes are the cell's waste disposal system, and their dysfunction is an early event in PD pathogenesis.[83] Patientssuffering from Gaucher disease, a rare, autosomal­recessive storage disorder caused by lysosomal enzyme β­glucocerebrosidase (GCase) deficiency,[84] have an increased risk of parkinsonism,[85] which appears to be driven by a direct

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effect of GCase deficiency and lysosomal dysfunction on α­Syn aggregation.[86]

Measuring GCase activity in the CSF could be a useful biomarker in PD. PD[87] and DLB[88] patients were found to havesignificantly reduced GCase activity compared with neurological controls. A recent study showed that the combination ofGCase activity, oligomeric/total α­Syn ratio and age discriminates best PD from neurological controls.[89] However, in a Dutchcohort of de novo PD patients and healthy controls, there was a trend towards a reduction in CSF GCase activity.[90] Theusefulness of GCase as a potential biomarker in parkinsonian conditions needs to be evaluated in future studies that includeadditional neurodegenerative groups to PD.

'Omics' Approaches. The markers already discussed have been hypothesis driven based on pathophysiological studies,which have identified potentially deranged pathways in neurodegenerative diseases. The 'omics' techniques offer anunbiased approach of identifying biochemical pathways that are unexpectedly involved in neurodegeneration. Ultimately, theaim is to generate a list of candidate markers deserving further targeted studies.[91] The 'omics' approach results in unbiasedand systematic measurement of patterns of variations in genes (genomics), RNA (transcriptomics), proteins (proteomics) andsmall molecules (metabolomics). We have briefly discussed genomics and touched on metabolomics in previous sections,and we will now review proteomics in parkinsonian disorders.

Abdi et al [92] used a multiplex quantitative proteomic platform to find 72 altered proteins in PD compared with healthycontrols. Apolipoprotein H and ceruplasmin seemed to differentiate PD from healthy controls and from non­PD patients (ADand DLB). Eight of the proposed proteins were validated using a multianalyte CSF profile and showed good PD discriminatorypower compared with AD and healthy controls.[93]

Using surface­enhanced laser desorption/ionisation time­of­flight mass spectrometry (SELDI­TOF MS), Constantinescu et al[94] found a CSF proteomic profile consisting of four proteins (ubiquitin, β2­microglobulin and two secretographin 1fragments), which differentiated PD and healthy controls from atypical parkinsonian patients with an AUC of 0.8. Recently,Ishigami et al [95] were able to differentiate PD from MSA, even at the early stages, using their proteomic pattern (ie, thecombined set of many protein peaks), rather than a single peak. Multiple peaks differentiated MSA and PD from controlgroups, consistent with previous reports that a panel of potential biomarkers is essential to distinguish between diseasestates.[96]

Another recent study attempted to differentiate PD from PDD patients using proteomic technology. Six proteins wereidentified, but only serin­protease inhibitor Serpin A1 was verified using biochemical methods. Performing 2­D immunoblots,there was 100% specificity and 58% sensitivity for the test procedure.[97] Testing CSF obtained from PD, PDD patients andnon­demented controls using a gel­free proteomics mass spectrometry approach with isotope­labelled samples (iTRAQ) ledto the identification of 16 differentially regulated proteins, which could be potentially diagnostic markers.[98]

While proteomics studies have produced a number of interesting candidate markers, these are still in need of replication andfar from being established. It has also become clear that many of the protein expression changes seen so far representchanges that are common to several neurodegenerative diseases. Reliable detection of disease­specific changes most likelydepends on the development of more advanced techniques that allow for deeper analyses of the CSF proteome.

Imaging Markers. Even though imaging biomarkers are beyond the scope of this review, we would like to point out thatcombination of CSF and imaging markers can provide increased diagnostic accuracy compared with using either modalityalone. For example, Borroni and colleagues used mid­sagittal midbrain­to­pons atrophy in addition to CSF tau fragmentslevels to increase the discriminative power in identifying PSP from other neurodegenerative conditions.[66]

Discussion

The vast majority of the studies discussed are cross­sectional, retrospective and do not have pathological confirmation. Theaccuracy of the clinical diagnosis is uncertain, and the contribution of comorbidity to the clinical phenotype is unknown.

There is lack of standardisation both of preanalytical (sampling collection, handling and storage) and analytical (analysisexecution/sample processing) factors. For example, CSF contamination by blood can alter study outcomes in α­Syn and DJ­1assays. In addition, there is lack of assay standardisation; different assays can give different absolute concentrations of theprotein, making it almost impossible to use global reference limits and diagnostic cut­off points.

Furthermore, both disease groups and control groups are heterogeneous. The neurodegenerative groups differ in terms ofage, disease duration and severity. The control groups include a very small proportion of healthy controls and are mostly non­neurodegenerative neurological patients. However, some studies include patients with possible neurodegenerativeconditions, such as mild cognitive impairment or normal pressure hydrocephalus.

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Finally, there is lack of combination of different biomarker modalities, such as imaging and CSF markers.

A very promising study is the Parkinson's Progression Markers Initiative (PPMI), which aims to identify PD progressionmarkers and to better define subsets of PD patients.[99,100] It is a 5­year, multicentre, longitudinal study of drug­naïve PDpatients with early­stage disease, compared with healthy controls. Detailed motor and neuropsychological assessments,DaT­scan and CSF examinations are performed. There is strict standardisation of data acquisition, CSF collection andprocessing.[21]

Summary Points: CSF Biomarkers in Parkinsonism

Aβ42 has a role in predicting cognitive decline in Parkinson's disease (PD)

t­α­Syn: most promising marker; differentiates synucleinopathies from other neurodegenerative diseases and controlsbut is not specific

t­tau and p­tau: inconsistent data, can help differentiate PD from AD and can be useful in combination with othermarkers

NF­L: useful in differentiating PD from atypical parkinsonian conditions

4R­tau: possible marker of disease progression in PSP

DJ1: potential role in discriminating MSA from PD

Oxidative stress/inflammatory/metabolic markers: promising initial results, requiring further validation

Future Developments for the CSF Field in Parkinsonism

We think that several hypothesis­driven biomarkers are going to be investigated at the same time using multiplex platforms.The proteomics field is likely to expand and gain in analytical sensitivity, resulting in the identification of more candidatemarkers, some of which may be unexpected and give new clues on disease mechanisms. There needs to be large,prospective and longitudinal cohorts with serial CSF examinations and pathological confirmation in as many patients aspossible. A very important issue to be resolved is the standardisation of protocols and improvement in quality controls in CSFanalysis. Finally, like in AD, it will likely be important to combine several CSF markers with other modalities, like imaging.

Accurate diagnosis of parkinsonian conditions should occur as early as possible, before too much irreversible neuronaldamage has accumulated. This is essential, especially with the emergence of potential disease­modifying drugs, which mustbe used to target the correct underlying pathology. There is promising progress in the development of an α­Syn imagingagent, using radio ligands that bind to α­Syn fibrils. This should enable the assessment of the distribution of brain α­Synduring life.[101]

Conclusion

Parkinsonian conditions, like most neurodegenerative diseases, have complex and dynamic interaction of several underlyingpathogenic mechanisms. A combination of biomarkers possibly from different modalities in large, longitudinal cohorts mightbe required for early diagnosis and accurate disease prognosis.

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Acknowledgements

We would like to thank Dr Janice Holton for kindly providing the pathological images.

Contributors

All authors contributed to the design of this review. NM drafted the manuscript, and HZ and AJL revised it.

Funding

NM is funded through the PSP association. HZ is funded through the Leonard Wolfson Experimental Neurology Centre.

Competing interests

None.

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Provenance and peer review

Not commissioned; externally peer reviewed.

J Neurol Neurosurg Psychiatry. 2014;85(10):1065­1075. © 2014 BMJ Publishing Group